The long-term objective of this project is to develop an understanding of the acute modulation of dopamine (DA) transporter (DAT) function by amphetamine (AMPH) and other DAT substrates. DA signaling at CNS synapses regulates a variety of cognitive, emotional and behavioral functions. Abnormalities in the dopaminergic system have been implicated in a number of psychiatric and neurological disorders, including drug addiction, schizophrenia and Parkinson's disease. AMPH induces reverse transport of DA, thereby increasing extracellular DA levels and leading to its psychostimulant effects. Exciting new experiments suggest that AMPH has unique interactions with the DAT. Thus, in contrast to DA, AMPH stimulates CaMKII activity, causes DAT associations with the SNARE protein syntaxin 1A, and induces DAT channel-like activity that results in rapid (msec) bursts of DA efflux. This project will combine molecular and biochemical approaches with biophysical and real-time imaging methodologies to characterize AMPH regulation of DAT function both in heterologous and neuronal preparations. The key issues to resolve include how AMPH differs from DA in its ability to induce DAT-mediated reverse transport of DA and DAT channel-like activity and whether DAT N-terminal phosphorylation and subsequent protein associations are essential for these actions of AMPH. Our experimental plan links the AMPH-induced functional regulation of DAT to stimulation of cytoplasmic signals that regulate the association of DAT and other proteins, thereby modulating DA efflux. The proposed studies address the following Specific Aims: 1) To identify differences in the abilities of AMPH and other DAT substrates to stimulate cytoplasmic signals leading to DA efflux. 2) To identify elements in the N-terminus that regulate the action of AMPH and to assess the impact of phosphorylation on the ability of AMPH to induce DA efflux. 3) To determine whether kinase activity and phosphorylation of DAT N-terminal serines regulate the interaction between DAT and syntaxin 1A, thereby modulating AMPH-induced DA efflux. The goal is to generate an experimentally-based model advancing our understanding of acute AMPH regulation of the DAT and to discover novel pathways and molecules that may contribute to disrupted dopaminergic signaling in disease states such as addiction.